We are developing human parainfluenza viruses (HPIVs) and avian paramyxoviruses (APMVs) as vaccine vectors for human use against highly pathogenic emerging viruses, using HPIV3 and Newcastle disease virus (NDV) as proof of principle. This strategy takes advantage of the natural respiratory tract tropism of HPIVs and NDV to provide respiratory administration and stimulate strong systemic immune responses as well as local mucosal immunity that is important for restricting pathogens that infect and are spread via the respiratory tract and conjunctiva. In primates, the replication of the HPIVs and NDV is limited to the respiratory tract, reducing safety concerns compared to systemic viruses. We previously showed that a single immunization of African green monkeys with a PIV3-based vector (an attenuated chimera of HPIV3 and BPIV3 called B/HPIV3) expressing the SARS-CoV spike S glycoprotein induced immunity sufficient to prevent shedding by a high-dose IN/IT challenge of SARS-CoV. We also previously showed that a single immunization of rhesus monkeys with HPIV3 expressing glycoprotein GP of Ebola virus (EBOV) was 78% effective in preventing mortality from an EBOV challenge, and two doses provided sterilizing immunity and protection in all of the animals. During the past two years, we further evaluated HPIV3 as a vector for glycoprotein GP of EBOV using the aerosol route of administration, compared with our previous method of nasal drops. These studies have been in collaboration with Dr. Alexander Bukreyev of the University of Texas Medical Branch, Galveston. In rhesus monkeys, a single dose of HPIV3-EBOV-GP vaccine induced levels of EBOV-specific IgA, IgG, and neutralizing antibodies in serum and mucosal specimens that equaled or exceeded that induced by the same vaccine given by nasal drops. The serum antibody responses induced by this single dose was substantially and significantly greater in abundance and avidity than that induced by a single dose of an alphavirus replicon vector expressing EBOV GP evaluated in parallel (administered intramuscularly), and was equivalent to that induced by two doses of the alphavirus vaccine. We also measured EBOV-specific CD4+ and CD8+ T lymphocyte responses in the lungs, blood, and spleen. This showed that the magnitude of these responses to the HPIV3-EBOV-GP vaccine were dramatically greater in the lungs than in the blood and spleen. The majority of lung CD8+ T cells were positive for two or more markers of activation (CD107a, IFNg, IL-2, and TNFa), indicative of efficient activation and a polyfunctional response. Compared to the HPIV3-EBOV-GP vector, the T cell responses to the alphavirus vector were comparable in magnitude in the blood and spleen, whereas in the lung they were much lower and less polyfunctional. When immunized rhesus monkeys were challenged with an otherwise lethal dose of EBOV delivered intramuscularly, a single aerosol dose of HPIV3-EBOV-GP provided complete protection, with no detectable challenge EBOV replication. We have manufactured a lot of HPIV3-EBOV-GP following Good Manufacturing Practices, using substrates and reagents suitable for human use. This experimental vaccine will be evaluated in adult volunteers, administered by intranasal spray, for safety and immunogenicity in studies beginning in Q3 2015; results will not be available this reporting year. We also previously created a version of HPIV3-Ebola-GP in which the HPIV3 F and HN genes were deleted, leaving Ebola GP as the sole viral surface antigen. The removal of the HPIV3 surface proteins should make this virus less sensitive to pre-existing immunity against HPIV3. In guinea pigs, this virus was very highly attenuated, with essentially no virus shedding, but a single immunization induced sterilizing immunity against Ebola challenge. In the past year, in collaboration with Dr. Alexander Bukreyev, this virus was evaluated in NHP. It induced a 6-8-fold lower titer of EBOV-specific serum neutralizing antibodies compared to the parental vector containing HPIV3 F and HN, reflecting its greater attenuation, but it conferred an equivalent level of protection against an otherwise lethal intramuscular challenge with EBOV. This virus also has been prepared as clinical trial material for pending clinical evaluation in adults. We previously showed that NDV is highly restricted in non-human primates due to host range differences. Indeed, most immunized animals did not detectably shed NDV, and direct analysis of lung tissue revealed very low levels of replication. NDV-based vectors expressing protective antigens of Ebola virus, SARS-CoV, or HPAIV were immunogenic and protective (the latter property was evaluated for SARS and HPAIV) against the respective pathogen in non-human primates, although two doses were used due to the high level of attenuation of the vector. We mostly have used the mesogenic (intermediate virulence) NDV strain Beaudette C as a vector. However, mesogenic NDV was classified as a Select Agent several years ago, which complicates and limits its use. Low-virulence (lentogenic) strains of NDV are not Select Agents but may be suboptimally immunogenic due to a greater restriction of replication. Therefore, over the past several years, we have worked with Dr. Siba Samal of the University of Maryland to modify the mesogenic BC strain with the goal of removing the characteristics that classify it as a Select Agent (specifically, the presence of a polybasic furin cleavage site in the fusion F protein and an intracerebral pathogenicity index ICIP of &#8805;0.7) while maintaining or increasing its effectiveness as a vaccine vector. For example, we changed the multibasic cleavage site sequence of the F protein to the dibasic sequence of lentogenic (avirulent) strain LaSota. Additionally, the BC F and HN proteins were modified in several ways to reduce virulence while maintaining or even enhancing virus replication. This included replacing the BC HN gene with that of La Sota, and replacing segments of the BC F protein with their counterparts from a velogenic (highly virulent) African AKO NDV strain. These modified BC-derived vectors and the control LaSota strain previously were engineered to express the hemagglutin (HA) protein of H5N1 HPAIV as a test foreign antigen. In general, the modified BC-based vectors expressing HA replicated better than the LaSota/HA control, and expressed higher levels of HA protein. Pathogenicity tests indicated that all of the modified viruses were highly attenuated in chickens. Selected vectors were evaluated in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04, and were highly protective. This identified the presence of residues 271-330 from the F protein of NDV AKO as being particularly important for high efficacy. In further studies, the presence of AKO F residues 331-309 also appeared to enhance fusion and replication, whereas the presence of AKO F residues 391-450 inhibited fusion and replication. These strains also were very highly attenuated such that they are not classified as Select Agents. These findings suggest that these modifications will provide a highly attenuated and safe vaccine vector with enhanced replication, expression, and protective efficacy. NDV strains fall into three different genome-length groups due to an insert in the N gene or the P gene, the latter affecting amino acid coding. We evaluated the potential significance of these inserts by placing them into infectious virus. This showed that the inserts had no evident effect on gene expression, protein function, or replication in vivo, but in the majority of cases were associated with a decrease in virulence in standard assays. Thus, the appearance of these inserts in nature appears to be associated with increasing attenuation by an unknown mechanism.